One important type of ionization for biomolecules is ionization by matrix-assisted laser desorption and ionization (MALDI). This process ionizes the analyte biomolecules from mixtures with molecules of a matrix substance on sample supports. The ratio of analyte molecules to matrix molecules is around one to ten thousand. Hundreds of samples can be applied to a sample support using pipetting robots which are available for this purpose. Transporting the samples on the sample support into the focus of a UV pulsed laser takes only fractions of seconds; as much time as is ever needed is available for the analysis of this sample (until the sample is completely used up). This sets MALDI very favorably apart from electrospray ionization, which provides only very slow sample changing or, in conjunction with chromatography, restricts the analysis time to the duration of the chromatographic peak. MALDI is ideal for the identification of tryptically digested proteins which have been separated by 2D gel electrophoresis. The MALDI analysis of peptides which have been separated by liquid chromatography and applied to MALDI sample supports is also gaining ground (“HPLC MALDI”).
A disadvantage of MALDI, however, is that it only ionizes one ten thousandth of the analyte molecules. One attomol of an analyte substance, i.e., approx. 600,000 molecules, only produces around 60 analyte ions. The rest are not ionized; some of the remaining molecules are possibly contained in splashes of molten matrix substance and completely excluded from access to ionization, while a large proportion of the analyte molecules are simply not ionized in the laser desorption process.
Until now, matrix-assisted laser desorption has principally been carried out in high vacuum. Its starting point is a solid sample mixture prepared on a sample support. The sample consists mainly of small crystals of the matrix substance which are admixed in low proportions (only around one hundredth of one percent or less) with molecules of the analyte substance. These analyte molecules are embedded individually into the crystal lattice of the matrix crystals or are located in inter-crystal boundary surfaces. The samples prepared in this way are irradiated with short pulses of UV laser light. The duration of the pulses is roughly between two and ten nanoseconds. This produces vaporization clouds which contain both ions of the matrix substance as well as some analyte ions. Some of the analyte ions are already contained in the solid sample in ionized form, some are created directly in the explosive vaporization process in the hot plasma, and a third fraction is formed in the expanding cloud by proton transfer in reactions with the matrix ions.
Laser desorption, which was previously only used in high vacuum, has of late also been used at atmospheric pressure, simplifying the sample introduction but not, as yet, increasing the analytical sensitivity. This method is termed AP-MALDI.
This laser desorption at atmospheric pressure is characterized by the formation of a vapor cloud which arises when sample preparation material is vaporized in pulses by the laser light bombardment, and which can be entrained with the ambient gas. The vapor cloud initially consists only of matrix vapor with analyte molecules blown likewise into the gaseous phase. Only a very small fraction of the analyte molecules, in the order of one hundredth of one percent or less, are ionized. The matrix substance is similarly weakly ionized; in absolute figures, however, the matrix ions are in the majority many times over. This vapor cloud mixes in a thin boundary layer with ambient gas, but remains together for a long time. In the vapor cloud, the matrix ions can thus react further by collisions with analyte molecules, forming analyte ions. It is thus possible that more analyte ions are formed at atmospheric pressure than with MALDI in high vacuum, but this advantage is counteracted by the disadvantage that the ions from this more or less expanded vapor cloud are generated at atmospheric pressure and have to be transferred into the vacuum system of the mass spectrometer. The analyte ions which are lost as a result have, until now, been greater in number than the gain in additional analyte ions—if, indeed, this occurs at all.
A method of matrix-assisted laser desorption at atmospheric pressure has also been described in which it is not the laser desorption itself which brings about the ionization of the analyte molecules but the ionization is achieved in the subsequent ion/molecule reactions. The matrix substance thus no longer has to perform the task of ionizing the analyte molecules. For the desorption here it is possible to specifically use a substance for the matrix which decomposes into small gas molecules under laser bombardment and thus has only the three objectives of (1) binding the analyte substances firmly to the surface of the sample support, (2) absorbing the laser light, and (3) vaporizing in such a way that it decomposes, transferring the confined analyte molecules intact and isolated from each other into the gaseous phase. The chemical ionization is then undertaken, for example by forming primary ions by means of the electrons of a corona discharge (J. Franzen and C. Köster, DE 196 08 963; corresponding to GB 2 299 445 and U.S. Pat. No. 5,663,561). It is difficult to carry out the chemical ionization, however, since it is very difficult to get the gas cloud with the analyte molecules and the gas cloud with the ions for the chemical ionization to mix sufficiently intensively with each other in a short time.
The desorption of analyte molecules does not have to be carried out with laser light. It is also possible to desorb adsorbed molecules with shock waves, temperature shocks and especially with vacuum sparks, even if desorption with laser light is by far and away the easiest option.
MALDI in an ambient gas at a pressure of around 100 pascal has already been reported (A. N. Krutchinski et al., Rapid Comm. Mass Spectrom. 12, 1998, 508-518). A MALDI ion source which also operates in this pressure range but under a pressure surge of a gas introduced as pulses has been described in DE 199 11 801 C1 (G. Baykut).
Whenever the term “mass of the ions” or simply just “mass” is used here in connection with ions, it is always the “charge-related mass” m/z which is meant, i.e., the physical mass m of the ions divided by the dimensionless and absolute number z of the positive or negative elementary charges which this ion carries.
The term “desorption” here should be taken to mean all types of release of molecules from the solid phase into the gaseous phase, as is already the case with the term MALDI (matrix assisted laser desorption and ionization). It is irrelevant here whether the molecules to be desorbed are adsorbed naked on a surface or whether they are embedded in, or attached on, another material, of any type.
An “ion guide” here should be taken to mean a device by means of which ions can be intentionally transported from one location to another. This can be achieved aerodynamically or by systems of electric and magnetic electrodes or yokes, and particularly by rod systems or systems of apertured diaphragms to which the phases of an RF voltage are applied.